Characterization of a cryptic plasmid isolated from Lactobacillus casei CP002616 and construction of shuttle vectors based on its replicon.

The cryptic plasmid pLC2W was isolated from Lactobacillus casei CP002616. Nucleotide sequence analysis revealed that 4 putative open reading frames (ORF) were responsible for DNA replication. Four Escherichia coli-Lactobacillus shuttle vectors were constructed using different lengths of the pLC2W replicon to identify the shortest functional replicon. The length of the pLC2W replicon did not affect the stability of the plasmids. Green fluorescent protein (GFP) as a reporter was expressed successfully in several lactobacilli using our constructed vectors. The results suggested that the expression vectors pUE-F0GFP and pUE-F1GFP are potential molecular tools for heterologous gene cloning and expression in lactobacilli. Moreover, 2 plasmid-curing methods were used to eliminate pLC2W from L. casei. We detected no difference between L. casei CP002616 and L. casei CP002616 pLC2WΔ-IC (mutant strain cured by plasmid incompatibility method) in production of exopolysaccharide (EPS) or acid. However, EPS and acid production were both reduced in L. casei CP002616 pLC2WΔ-HT (mutant strain cured by high-temperature heat treatment method), demonstrating a difference between these 2 curing methods. Sequence analysis of pLC2W and plasmid curing data suggest that plasmid pLC2W is not involved in EPS synthesis.